COP and Clathrin-Coated Vesicle Budding: Different Pathways, Common Approaches Harvey T Mcmahon1 and Ian G Mills2

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COP and clathrin-coated vesicle budding: different pathways, common approaches Harvey T McMahon1 and Ian G Mills2

Vesicle and tubule transport containers move proteins and of these coated vesicles have now been purified: COPI- lipids from one membrane system to another. Newly forming (coatomer) and COPII-coated vesicles (where COP transport containers frequently have electron-dense coats. stands for coat protein complex) and clathrin-coated Coats coordinate the accumulation of cargo and sculpt the vesicles [2–6] (Figure 1). Coat components are needed membrane. Recent advances have shown that components of for generation of highly curved membrane areas, recruit- both COP1 and clathrin-adaptor coats share the same structure ment of cargo (and exclusion of non-cargo proteins/ and the same motif-based cargo recognition and accessory lipids), vesicle scission and uncoating factor recruitment. factor recruitment mechanisms, which leads to insights on Not all budding pathways lead to small vesicles, but the conserved aspects of coat recruitment, polymerisation and high membrane curvature of these transporters makes membrane deformation. These themes point to the way in them intrinsically more fusogenic than larger vesicle which evolutionarily conserved features underpin these structures like macro-phagosomes. There may be many diverse pathways. ways to bud a membrane but we would argue that producing a small vesicle that concentrates cargo mole- Addresses cules requires a coat. 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK Just as a SNARE-based fusion underpins the hypothesis e-mail: [email protected] 2Department of Oncology, University of Cambridge, Hutchison MRC of a common mechanism for all membrane fusion, so a Cancer Research Centre, Hills Road, Cambridge, CB2 2XZ, UK coat-based budding hypothesis has emerged as a common e-mail: [email protected] theme in vesicle budding. This review highlights conserved mechanisms underpinning diverse budding pathways and the differences that often reflect specificity and Current Opinion in Cell Biology 2004, 16:379–391 regulation. This review comes from a themed issue on Membranes and organelles Background to coated vesicle budding Edited by Judith Klumperman and Gillian Griffiths Clathrin-coated vesicles are named after the protein that Available online 20th June 2004 self-polymerises into a lattice around these vesicles as they bud from the plasma membrane, trans-Golgi net- 0955-0674/$ – see front matter work (TGN) and endosomes [7,8]. For all clathrin-coated ß 2004 Elsevier Ltd. All rights reserved. vesicles, clathrin is the central organiser (Box 1). It concentrates cargo adaptors, leading to a diverse protein and DOI 10.1016/j.ceb.2004.06.009 lipid load in the forming vesicle, and its polymerisation into a curved lattice stabilises the nascent membrane bud Abbreviations AP adaptor protein as it forms. Each clathrin molecule contains an N- Arf ADP ribosylation factor substrate terminal domain that has a b-propeller structure and binds ARH autosomal recessive hypercholesterolemia protein to peptide motifs between its blades [9]. This multi- COP coat protein complex bladed propeller allows for multiple protein interactions ER endoplasmic reticulum with various specificities [10], recruiting a versatile array GGA Golgi-localised, g-ear containing, ADP-ribosylation factor-binding protein of different cargo adaptors and membrane attachment Hrs hepatocyte growth factor receptor tyrosine kinase substrate proteins [11,12,13 ,14]. TGN trans-Golgi network The main cargo adaptor proteins characterised to date are the classical adaptor protein (AP) complexes, AP1, AP2, Introduction: The coated vesicle budding AP3 and AP4 [15–19] (Table 1). Most AP complexes hypothesis adapt and/or link clathrin to selected membrane cargo and Early electron microscopy studies of cells led to a vesicle- lipids, and they also bind accessory proteins that regulate transport hypothesis for protein trafficking. Vesicular coat assembly and disassembly (such as AP180, epsins transport intermediates bud from a donor organelle and and auxilin; see Table 2). ‘Alternative adaptors’ can also then fuse with an acceptor organelle. Budding intermedi- function in clathrin-mediated vesicle budding. These ates were initially identified by their electron-dense adaptors are sometimes called ‘monomeric adaptors’ ‘coats’ and were found on the plasma membrane and but this does not accurately describe some of the mem- intracellular organelles [1] (Figure 1). Three major classes bers of this group, which are dimeric. GGA adaptors www.sciencedirect.com Current Opinion in Cell Biology 2004, 16:379–391 380 Membranes and organelles

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Box 1 Definitions factors (Figure 1 and Table 1) [20,21]. GGAs and Hrs (hepatocyte growth factor receptor tyrosine kinase sub- Clathrin Clathrin is a trimer with a central hub domain from which extends strate) have similar but not identical domain compositions three legs ending in terminal (b-propeller/WD40) domains. This (Figure 1). By analogy with cargo binding to the GGA building block of a cage structure is known as a triskelion. During VHS domain, Hrs may also bind cargo, but this protein is polymerisation the legs on neighbouring triskelia twist around each targeted via its FYVE domain to endosomes. Arrestins, other and make a lattice. Recruitment proteins bind to these terminal domains and help to concentrate clathrin on membranes. epsins, disabled-2 and ARH (autosomal recessive Concentrated clathrin self-assembles [7]. hypercholesterolemia protein) can also be classified as alternative adaptors as they also link cargo and mem- Cargo adaptors Proteins that link cargo into the clathrin-coated pit branes to the clathrin lattice (Table 2) [11,13 ,14,22]. These may work in some cases alongside or indepen- Classical clathrin adaptors: AP1, AP2, AP3 and AP4 complexes each have four subunits: two dently of classical adaptors to recruit their cargo into large, one medium and one small, which are believed to be stably budding vesicles [23 ]. Since multiple adaptors are found associated in the cell. AP1 is involved in protein sorting from the TGN in individual coated pits, it is not necessary for all adaptors and endosomes; AP2 traffics from the plasma membrane; AP3 to have a direct clathrin interaction. traffics to lysosomes and AP4 is found on vesicles in the vicinity of the TGN [19]. COP coats come in two very different flavours, belying Alternative clathrin adaptors the homologous nomenclature (Figure 1 and Table 1). Monomeric and dimeric cargo/clathrin adaptors like GGAs and Hrs COPI coat components have sequence homology to cla- Accessory proteins thrin AP complex proteins [24–28]. Just as clathrin and Clathrin recruitment, membrane bending and scission molecules. This group can cover all proteins involved in clathrin-mediated adaptors come together to form clathrin-coated vesicles, endocytosis except clathrin. But the definition is more usefully so the two subcomplexes of COPI coats come together to applied when it neither covers clathrin nor cargo adaptors. recruit cargo into buds [28]. COPI coat components traffic COP coat subcomplexes primarily from the Golgi to the endoplasmic reticulum There are seven COPI subunits (a, b, b’, d, g, e and z) divided into two (ER) and between Golgi cisternae. subcomplexes: the F subcomplex composed of b, d, g and z-COPs, of which the b and g subunits display homology to AP complex COPII-coated vesicles traffic from the ER to the Golgi. appendage domains, and the B subcomplex, within which the a and b’ subunits contain WD40 repeats akin to clathrin terminal There are again two subcomplexes (Figure 1 and Table 1) domains [28]. that come together to recruit cargo into buds. In the clathrin-like subcomplex of COPI coats (the B-subcom- COPII coat proteins can also be considered as subcomplexes consisting of Sec13/31p and Sec23/Sec24p. Both Sec13/31p have plex) and in the Sec13/31p subcomplex of COPII coats WD40 repeats akin to clathrin whereas Sec23/24p functionally there are b-propeller domains (made from WD40 repeats) resemble the AP complexes in a cargo recruitment capacity. [29] that form a protein interaction face just like the b- Motif domains propeller clathrin terminal domains. COP coats are there- Generally regions lacking tertiary structure in proteins containing fore also multisubunit protein complexes, whose compo- short sequence motifs that serve as binding sites for ligand. This is a nents select cargo, drive membrane curvature and vesicle generic term that can be applied to all proteins with similar domains budding (Table 1). We will now look at some common- across the proteome and stands in contrast to ‘structured domains’. It is likely that many of these domains have a limited structure. alities in these diverse vesicle budding pathways. Conservation of coat recruitment and (Golgi-localized, g-ear containing, ADP-ribosylation- membrane curving mechanisms factor-binding proteins) are associated with TGN clathrin A long-recognised commonality between COPI, COPII coats and bind cargo, membranes, clathrin and accessory and many clathrin budding pathways is that small

(Figure 1 Legend) Structural and functional homology between COP1 coats and clathrin-adaptor coats. (a) The ‘classical’ AP2 clathrin adaptor with its four subunits forms a complex that links cargo recruitment to clathrin. Clathrin is a trimer and each terminal domain is represented as being bound to a b2 adaptin appendage and hinge domain. The other heterotetrameric AP complexes, AP1, AP3 and AP4, have the same overall structure. (b) The COPI F-subcomplex probably has a similar topology to the AP complex and the B-subcomplex is likely to be the functional equivalent of clathrin. (c) Likewise, COPII has two subcomplexes, Sec23/24 and Sec13/31. (d) Alternative clathrin adaptors GGAs and Hrs are like the large subunits of AP complexes in that they bind to cargo, clathrin and membranes. Hrs is specifically recruited to endosomes via its FYVE domain, while GGAs are membrane recruited via Arf binding just like the COPI F-subcomplex and AP1, AP3 and AP4 clathrin adaptor complexes. GGA has been shown to bind cargo via its VHS domains and both GGA and Hrs also have ubiquitin binding regions. Also, both bind to clathrin, but only GGAs have the appendage domain necessary for accessory protein recruitment. PDB codes: AP2 core complex 1GW5; Clathrin terminal domain 1BP0; b2-appendage 1E42; a-appendage 1B9K; COP1 g-appendage 1R4X; sec23/24 1M2V; Hrs VHS-FYVE 1VDP; VPS27 UIM-ubiquitin 1Q0W; GGA1 appendage 1NA8; GGA3 VHS 1JPL; GGA GAT-Arf 1J2J. We acknowledge the kind permission of Pietro DeCamilli, Randy Schekman and Lelio Orci to use the following electron micrographs of budding vesicles from liposomes: clathrin-coated vesicle [58] (reproduced with permission from Elsevier); COPI vesicle [59] (copyright 1998 National Academy of Sciences, USA); COPII vesicle [60] (reproduced with permission from Elsevier). Scale bar for EM images of vesicle budding is 100 nm. www.sciencedirect.com Current Opinion in Cell Biology 2004, 16:379–391 382 Membranes and organelles

Table 1

Coat subunit functions and domains Colour coding: blue text, AP1/GGA-associated proteins; red text, AP2-associated proteins; purple text, COPI-associated proteins; green text, COPII-associated proteins. Adaptor/coat Salient features Peptide motifs Functions

Clathrin Endocytosis, sorting from TGN to endosomes, sorting from early to late endosomes Heavy chain Subunits polymerise into a triskelion; Binds LLDLD type 1 clathrin motif Recognition of peptide motifs through atomic structures of several and PWxxW type 2 clathrin motif. the b-propeller formed by WD40 fragments reveal a-zigzag repeats (terminal domain). Binding repeats and a partners include auxilin, amphiphysin, b-propeller terminal domain. epsin and AP180. The triskelial arms define skeleton of the clathrin coat. Light chain: LCa/b Interacts with Hsc70, calmodulin Regulates clathrin self-assembly and the central helical/coiled coil into polyhedral lattices. domain of HIP1/HIP12. AP1 adaptors TGN-endosome sorting g Large AP complex subunit with Binds DFGxØ and DFxDF motifs. Membrane binding via Arf1. truncated appendage domain. Recruitment of accessory factors to AP1 complexes e.g. EpsinR. b1 Clathrin box motif in hinge: LLNLD. Membrane binding via Arf1. Binds dileucine cargo motifs: Binds to clathrin via hinge domain. [DE]xxxL[LI]. Binds accessory proteins via appendage domain. Cargo recognition. m1 (A/B) Binds YxxØ cargo motifs. Cargo recognition. Membrane interaction. s1 s1 sequence is weakly related Binds [D/E]xxx[L/I] cargo motifs Stabilises the AP core complex to the N-terminal portion of m. in complex with the m subunit by mediating interactions between subunits. AP2 adaptors Plasma membrane endocytosis aA/C Atomic structure of the appendage Binds DxF, FxDxF and WVxF motifs Membrane binding. and trunk domain are known. Recruitment of accessory proteins e.g. Epsin1 b2 Atomic structure of the appendage Clathrin box motif in hinge: LLNLD. Binds to cargo dilucine motifs. and trunk domain are known. Binds cargo motif: [DE]xxxL[LI]. Binds clathrin via hinge domain. Binds accessory proteins via appendage domain. m2 Atomic structure of m2 and of Binds YxxØ in cargo and Membrane interaction. interactions with YxxØ sorting WVxF in stonin 2. Cargo recognition. motifs is known. Also interacts with accessory proteins such as stonin 2. s2 s2 sequence is weakly related Stabilises the AP core complex to N-terminal portion of m. by mediating interactions between subunits. AP3 adaptors Melanosome biogenesis d Appendage domain and trunk Like binds to accessory proteins via domains conserved. the appendage domain, but these have not been identified as yet. b3 (A/B) Appendage domain and trunk Clathrin box motif in hinge: LLDLD. Binds clathrin. domains conserved. Binds cargo motif: [DE]xxxL[LI]. Appendage domain with conserved ligand binding pocket likely to bind to accessory proteins. Cargo recognition. m3 (A/B) Binds cargo motif YxxØ. Cargo recognition. s3 (A/B) Binds [D/E]xxx[L/I] cargo motifs Stabilises the AP3 complex. in complex with the m subunit AP4 adaptors Basolateral sorting/ TGN-endosome sorting

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Table 1 Continued

Adaptor/coat Salient features Peptide motifs Functions

e Membrane binding via Arf1. Conserved appendage domain so likely to recruit accessory proteins. b4 No obvious clathrin box Binds the neuronal protein tyrosine phosphatases PTP-SL and PTPBR7. Has conserved appendage domain. m4 Binds YxxØ weakly and also Cargo recognition. interacts with other non-classical cargo motifs like DLYYDPM s4 Stabilises the AP4 complex. GGAs TGN-endosome/lysosome sorting GGA-1 Multidomain alternative adaptors Clathrin box motif in hinge: LLDDE. Clathrin binding. Recruitment of with VHS, GAT, hinge and Appendage domain binds accessory factors such as rabaptin-5 appendage domains. DFGxØ motifs. and p56 by appendage domain. VHS domain binds DxxLL motifs. Cargo recognition (e.g. Ci-M6PR) WNSF sequence in the hinge is via VHS domain. bound by the AP1 g appendage. Membrane recruitment via Arf binding to GAT domain. Ubiquitin binding via GAT domain and thus potentially binds ubiquitinated cargo. GGA-2 Clathrin box motifs in hinge: Same as for GGA-1 LIDLE and LLDLL. Other interactions are same as for GGA-1. GGA-3 Unidentified clathrin-binding Same as for GGA-1 motif and other interactions are same as for GGA-1. COPI Retrograde transport from the Golgi F-subcomplex: bdcf to the ER, maintenance of Golgi B-subcomplex:ab0e integrity Arf1 Small GTPase; Ras family. Recruitment of COPI coatomer to membranes in a GTP dependent manner. aCOP / Ret1p WD40 repeats (b-propeller domain). Binds KKxx, KxKxx motifs. Recruitment of cargo and accessory factors (eg Dsl1p). bCOP Binds Arf1, and has weak Binds to diacidic cargo motifs. sequence identity to b-adaptin. Has appendage domain Recruitment of cargo and accessory like large AP subunits. factors via appendage domain but these have not been identified. b0COP / Sec26p WD40 repeats (b-propeller). Binds KxKxx motif. Recruitment of cargo. gCOP / Sec21p Binds members of the p24 family. Recruitment of cargo. Has appendage domain like Recruitment of cargo and accessory large AP subunits. factors (ArfGAP) via appendage domain. dCOP / Ret2p Weak sequence identity Binds WxxxW motif in the Binds accessory protein via to m-adaptin. acidic domain of Dsl1p. acidic tryptophan motif. eCOP / Sec28p May stabilise the complex (point mutations are often lethal in yeast). zCOP / Ret3p Weak sequence identity Stabilises the interaction between to s-adaptin. b-COP and g-COP. ARFGAPs Glo3p GTPase activating protein for ARF. Activates Arf GTP hydrolysis to Binds b0 WD40 domain promote coat disassembly. and g-COP appendage domain. ARFGEFs GEF for ARF Gea1p/Gea2p

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Table 1 Continued

Adaptor/coat Salient features Peptide motifs Functions

COPII Protein export from the ER hSar1p Small GTPase of the Ras family. Recruits coat components to membranes in a GTP-dependent manner. hSec13p/hSec31p Both subunits have WD40 Not identified as yet. Induces coat polymerisation. repeats (b-propeller domains). hSec23p/hsec24p Sec23 is a Sar1 GTPase Sec24 binds DxE, YNNSNPF Has GAP activity for Sar1. activating protein and Sec24 and Lxx[L/M]E motifs. Cargo recognition and membrane binds cargo. curvature selection. hSec12p Has a GEF domain. ER localized GEF and thus leads to the recruitment of Sar1 and COPII coat components to the ER. hSec16p Forms a ternary complex Stabilises Sec23/24 complex and with Sec23/24p in vitro. stimulates vesicle budding. This table is referenced in full and updated online at: http://www2.mrc-lmb.cam.ac.uk/groups/hmm/adaptors/Table1.htm.

G-proteins are required for membrane recruitment Coat polymerisation leads to membrane [18,20,30,31]. COPI components and clathrin adaptors curvature (AP1, AP3, AP4 and GGAs) are recruited by Arfs (ADP Coat assembly may also lead to membrane budding. ribosylation factor substrates), whereas COPII depends Clathrin, in the absence of membranes and other proteins, on the Arf-related protein Sar1. An amphipathic helix is self-assembles into a coat with a similar curvature to coated extended from the N terminus of these G-proteins in the vesicles. The COPII membrane-associated sec23/24 com- GTP-bound forms [32]. It is likely that this amphipathic plex has an intrinsic curvature like that found in COPII helix lies on the surface of the membrane between the vesicles and thus by polymerisation this curvature will be lipid head groups and, when concentrated at the bud favoured [36]. The distinction between the structural site, either drives or facilitates membrane curvature curvature of a clathrin lattice or Sec23/24p and the forces like epsin [13]. The exception to Arf recruitment is required to impose curvature on a membrane are well clathrin-AP2 budding from the plasma membrane, illustrated by the recent study of BAR domain proteins. where PtdIns(4,5)P2 binds directly to AP2 and other The crystal structures of the S. cerevisiae Sec23/24–Sar1 accessory proteins and epsins help to generate mem- complex and the BAR domain of Drosophila amphihysin brane curvature. This difference may be due to different are both concave or ‘banana-like’ in appearance with topological constraints applying to the generation of positively charged amino acids providing an electrostatic curvature from a relatively flat membrane as opposed interface with the membrane. A protein will curve mem- to a positively curved membrane, and also to the rigidity branes if the difference in the energy of binding to curved of this bilayer. versus flat membranes is greater than the energy required for membrane deformation. The BAR domain of Droso- Coat polymerisation leads to cargo phila amphiphysin induces membrane deformation, and concentration mammalian amphiphysin 2 binds clathrin promoting poly- COPI and COPII coats all have the equivalent of a merisation in vitro. Give the association of Sec23/24–Sar1 clathrin and an adaptor subcomplex (Figure 1). These complex with a clathrin-like COPII subcomplex (Sec13/ subcomplexes can be purified and in each case one 31p-containing WD40 terminal-like domains) perhaps it subcomplex binds to membranes and the other associates would be timely to investigate whether Sec23/24–Sar1 is with the first to promote polymerisation of the coat. The both a sensor and inducer of membrane curvature. outer sheath, or clathrin-equivalent, can in all cases bind either directly or indirectly (via adaptors) to cargo using Just because the coat preferentially adopts a given cur- conserved b-propeller domains [33,34] (Table 1). The vature this does not mean that coat polymerisation pro- self-polymerisation of clathrin has been studied in great vides enough energy for curving membranes and that detail using cryo-electron microscopy [7] and it is this additional factors will not also promote this curvature. We oligomerisation that leads to the presentation of a con- have already proposed that small G-proteins will aid centrated field of adaptor/cargo binding domains (the b- membrane bending, and for clathrin-AP2 vesicles, epsin, propeller domains). Self-oligomerisation of clathrin is also amphiphysin and endophilin may also help to drive the promoted by b-propeller binding proteins [13,14,35]. membrane curvature in conjunction with clathrin poly- Thus coat assembly and cargo recruitment are dependent merisation (Table 2) [13,35,37]. Thus coat assembly on each other. may facilitate membrane curvature in two ways: first,

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Table 2

Accessory proteins involved in clathrin and COP coat formation. Colour coding: blue text, AP1/GGA-associated proteins; red text, AP2-associated proteins; purple text, COPI-associated proteins; green text, COPII-associated proteins. Accessory factor Salient features Peptide motifs Functions

Amphiphysin All forms have an N-terminal AP2 a-adaptin appendage Induces/senses membrane curvature. BAR domain (a dimerisation, lipid- binding motifs: FxDxF and DxF. Recruits dynamin to membranes. binding and curvature-sensing module). Clathrin b-propeller binding Promotes clathrin polymerisation All mammalian forms have a motifs: LLDLD and PWxxW. on membranes. C-terminal SH3 domain that SH3 domain binds to PSRPNR binds dynamin. motif in dynamin. The non-muscle splice forms have an insert that binds clathrin and the AP2 complex. AP180/CALM N-terminal ANTH domain AP2 a-adaptin appendage binding Recruits clathrin and promotes

binds PtdIns(4,5)P2. motifs: FxDxF and DxF motifs. polymerisation on membranes C-terminal region binds clathrin CALM has DxF and NPF motifs containing PtdIns(4,5)P2. Regulates and the AP2 complex. (bind to EH domains). vesicle size. Clathrin binding motifs are not well defined. ARFGAP GTPase activating protein for The peptide motif in ARFGAP has Activates ArfGTP hydrolysis to ARF. Binds bWD40 and g-COP not been described but is likely promote coat disassemby appendage. to resemble that found a and g appendage binding partners. ARFGEF GEF for Arf. Converts ArfGDP to ArfGTP. ARF1 Small G-protein. GTP binding motifs G1 to G4: Helps to recruit AP1, AP3, AP4, GGA ARF1 G1(GKT); G2(T); G3(VGG); and COPI F-subcomplex to G4(NKQD). membranes in a GTP-dependent Adaptor complexes and COPI manner. subunits are effector molecules. ARH1/2 N-terminal PTB domain binds to PTB domain recognizes NPVY An alternative cargo adaptor. May inositol lipids and receptor tails. motif in LDLR. also be found in combination with Binds to NPXY motifs and AP2. Clathrin b-propeller binding other adaptors. motif: LLDLE. Also binds b2-adaptin (motif not defined). b-arrestin Similar to visual arrestin whose Clathrin b-propeller binding An alternative cargo adaptor that structure has been solved. This motif: LIEFE. helps recruit clathrin and AP2 in domain binds inositol lipids and AP2 b2-adaptin appendage binding G-protein coupled receptor G-protein coupled receptors. motif: IVFEDAFR with the arginine endocytosis. A C-terminal extension binds being essential and corresponding clathrin and the AP2 complex. to R-394 in b2-arrestin. Auxilin Binds clathrin and contains a AP2 a-adaptin appendage binding Involved in uncoating of DnaJ domain and is a cofactor motifs: DPW and WDW. clathrin-coated vesicles. for Hsc70. Clathrin b-propeller binding motifs: DxF and DLL. DnaJ domain has a HPDK signature motif conserved across all auxilins and is required for Hsc70 binding and stimulation of its ATPase activity. Dab2/Doc2 N-terminal PTB domain binds to PTB domain recognises FxNPxY An independent cargo-specific adaptor

PtdIns(4,5)P2 and receptor tails. motif in the LDL receptor. that also binds to the AP2 complex. NPXY motifs and AP2. AP2 appendage binding motifs: FxDxF and DxF. Dsl1p Interacts with a-andd-COP and Contains WxW and WxxxW motifs Integrates cargo with the ‘clathrin-’ ER resident protein Tip20p. in an acidic central domain and ‘AP-like’ subcomplexes of COPI Dynamins Large GTPase that self-oligomerises GTP binding motifs G1 to G4: Vesicle scission on GTP hydrolysis. into a helix on negatively charged G1(GKS); G2(T); G3(DLPG); G4(TKLD). membranes. Changes conformation on GTP hydrolysis. Endophilins N-terminal BAR domain (a dimerisation, SH3 domain binds to PPxRP motif Induces/senses membrane curvature. lipid-binding and curvature-sensing in dynamin and PxRPP motif in Recruits the lipid phosphatase, module). C-terminal SH3 domain that synaptojanin. synaptojanin, to membranes, and binds synaptojanin and dynamin. thus is involved in uncoating. www.sciencedirect.com Current Opinion in Cell Biology 2004, 16:379–391 386 Membranes and organelles

Table 2 Continued

Accessory factor Salient features Peptide motifs Functions

Epsin1 N-terminal ENTH domain of epsin1/ AP2 a-adaptin appendage binding Induces membrane curvature in

EpsinR epsinR binds PtdIns(4,5)P2 and PtdInsP motifs in epsin1: DPW. conjunction with promoting clathrin respectively and promotes initial AP1 g-adaptin appendage binding polymerisation. Epsin1 may bind membrane curvature. Epsin1 has three motifs in epsinR: DFxDF. ubquitinated cargo via its UIMs. ubiquitin binding motifs (UIMs). Both Clathrin b-propeller binding motif epsin1 and epsinR have a in epsin1 is LMDLADV and LVDLD, clathrin/adaptor binding domain. and is DLFDLM in epsinR. Eps15 EH domain binding motifs: three copies of NPF motif in epsin1. The ubiquitin binding signature is ExxxLxLAxAxS[K/R/Q]. Eps15 N terminus has three EH domains AP2 a-adaptin and b-adaptin Located at the edges of clathrin-coated Eps15 that bind the NPF motifs in epsin1. appendage binding motifs in pits. Thought to organise epsins and Also has a clathrin/adaptor binding epsin1: multiple DxFs other molecules at the leading edge domain. of coated pit formation. HIP1/HIP1R N-terminal ANTH domain binds AP2 a-adaptin appendage binding Thought to provide a link between

PtdIns(4,5)P2. C-terminal region motifs in HIP1: FxDxF and DxF. actin and clathrin nucleation. binds clathrin, the AP2 complex Clathrin b-propeller binding and actin. motif in HIP1: LMDMD. Clathrin b-propeller binding motif in HIP1R: LIEIS. Intersectin/ Has multiple EH and SH3 domains Binds proline-rich motifs in ligands Links actin polymerisation to proteins DAP160 and a RhoGEF domain. Binds through its SH3 domains and involved in clathrin-coated vesicle N-WASP, cdc42, dynamin and NPF motifs in ligands via its formation. SNAP-25 among other proteins. EH domains. P56 Binds to g-adaptin and GGA GGA/AP1 g-adaptin appendage Unknown function appendage domains in vitro and binding motifs: DFxxF. co-localises with GGAs in vivo. PACS1 Binds to AP1 and AP3 complexes AP1 complex interaction via Recruits cargo containing and acidic cluster motifs in cargo ETELQLTF sequence (with mu diacidic motifs. such as M6PR, Nef and furin. and sigma subunits). Sar1p Small G-protein. GTP binding motifs G1 to G4: Binds to membranes when occupied G1(GKT); G2(T); G3(DLGG); G4(NKID). by GTP and recruits COPII coat components. GTPase activity implicated in coat disassembly upon vesicle budding. Stonins (1/2) Binds Eps15, AP2 and AP2 a-adaptin appendage Assists AP2/synaptotagmin synaptotagmin. binding motif: WVxF. recruitment Eps15 EH domain binding motifs: two copies of NPF in stonin2. Synaptojanin Phosphoinostide 50-phosphatase AP2 a-adaptin appendage binding A lipid phosphatase that is recruited and SacI phosphatase domain. motifs: FxDxF and WxxF are found to coated vesicle via endophilin. By Binds to the AP2 complex and in the 170kDa splice form and may dephosphorylation of inositol lipids this has a proline-rich domain that binds be involved in the recruitment of will weaken the attachment of coat to endophilin and amphiphysin. synaptojanin to coated pits. proteins and thus this protein is likely involved in uncoating. Synaptotagmin1 Calcium sensor for exocytosis. WHxL motif is essential for May integrate exo-and endocytosis Gets endocytosed by binding to plasma membrane association. in nerve terminals the AP2 complex and to stonin2. Syndapin/Pacsin C-terminal SH3 domain. Regulates clathrin-coated Binds N-WASP and dynamin. vesicle motility Hsc70 ATPase that binds to auxilin. Clathrin coat release. Hsc70 is also called the ‘uncoating ATPase’. g-synergin Has EH domain that binds to AP1 g-adaptin appendage Unknown function in NPF motifs in SCAMP. Also binds binding motif: DFxDF. clathrin-AP1 trafficking. to the AP1 complex. Some of the accessory proteins are alternative adaptors while others are involved in organising cargo adaptors, sculpting the vesicle and in vesicle scission and uncoating. This table is referenced in full and updated online at: http://www2.mrc-lmb.cam.ac.uk/groups/hmm/ adaptors/Table2.htm.

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directly through the inherent curvature of the coat com- b-sandwich subdomain. These two subdomains are pre- plex, and second, indirectly through the recruitment of sent in most appendage domains, whereas g-adaptin and accessory factors capable of promoting curvature. the structurally-related GGA adaptors have only the b- sandwich subdomain [43–47].Atfirst it was thought that Structural and functional similarity between the platform subdomain contained the only ligand-bind- COPI and adaptor protein complex ing site, but ligand binding to both the b sandwich appendage domains subdomains of g and GGA appendages has prompted a The aforementioned similarities are largely mechanistic re-evaluation of the possibility that each subdomain has a but there is also structural similarity between COPI and distinct binding site for ligands. These studies are still clathrin-AP vesicle budding [38,39]. The large sub- ongoing in various laboratories. In an AP complex there units of adaptor complexes have domains called ears or are always two appendage domains and thus there may be appendages that, as shown by electron microscopy, are up to four independent protein interaction sites per AP attached to the main trunk of the AP complex by a flexible complex in addition to clathrin binding sites on the linker known as a hinge (Figure 1). The first appendage flexible hinge region (Figure 1). structure to be solved was from the AP2 a subunit [40,41,42]. It contained two sub-domains (Figure 2), a There is strong structural homology between AP complex platform with a-helical and b-sheet content and a support appendages and the g-COP appendage (Figure 2). We

Figure 2 Platform subdomain -sandwich subdomain β

γ appendage GGA1 appendage α appendage

β2 appendage GGA3 appendage γ-COP appendage Current Opinion in Cell Biology

The amazing structural similarity of appendage domains from COPI (g-COP), clathrin-AP complex (a-, b-andg-appendages) and GGAs (GGA1 and GGA3). Binding sites are highlighted with important residues and where available structures of bound peptides are shown in greater detail. In GGA and g-adaptin appendages the upper platform domain is absent. It is likely that this lower b-sandwich binding site is conserved in other appendages, alongside the platform binding site. Movies of individual appendage domain highlighting their binding sites are available at http://www2.mrc-lmb.cam.ac.uk/groups/hmm/adaptors/Appendages.html. PDB codes: a-appendage 1KY7, 1KYD; b2-appendage 1E42; COP1 g-appendage 1R4X; g-adaptin appendage 1GYU; GGA1 appendage 10M9; GGA3 appendage 1P4U. Drawings were made with AESOP (Martin Noble, unpublished work). www.sciencedirect.com Current Opinion in Cell Biology 2004, 16:379–391 388 Membranes and organelles

note by sequence homology that b-COP also has a con- concentrated by clathrin polymerisation. When the cla- served appendage. The presence of two subdomains thrin cage is disassembled and the adaptors dispersed, again points to the possibility of two ligand-binding sites these low affinity interactions will readily fall apart. Thus, on COP appendages. Indeed the tryptophan on the plat- this mode of interaction by COPs and clathrin coats is form subdomain that forms the central binding pocket well adapted for proteins that only need to interact during in a-adaptin is conserved in both b- and g-COP, and coat assembly. mutagenesis of g-COP abolished the interaction with ArfGAP [39]. This shows a structural conservation in Given the similarity of the appendage platform binding coat components and points to a common ancestral origin site of COP to the binding site on a-adaptin and the for coated vesicle budding mechanisms. It also shows a conservation of surrounding basic residues, it is likely that common function for appendage domains in recruiting the COP accessory protein binding motifs will be similar accessory proteins needed for the budding process. and may be of the form [D/E]x[F/W]. Looking through the ArfGAP2 sequence we find multiple copies of these Accessory proteins promote vesicle motifs. When a more precise idea of the motif identity is uncoating in COPI and clathrin-coated achieved, one will be able to search the protein database vesicles for other potential COP ligands and accessory proteins In cases where we know the functions of the appendage- for COP vesicle budding, as has previously been done for domain binding partners we can say that they are involved AP-dependent pathways. in congregating the proteins necessary to polymerise, invaginate and promote fission of the nascent vesicle Clathrin versus COPI coats (Table 2). For example, AP2 complexes bind to epsin1, COPI and COPII coats are used in the biosynthetic and which is involved in driving membrane bending in addi- recycling pathways between the Golgi and the ER, tion to promoting the polymerisation of clathrin [13]. whereas clathrin coats are found on endocytic and recy- The AP1 appendage domains bind a homologue of this cling pathways between the plasma membrane and the protein, epsinR, which also binds clathrin and membranes lysosomes or the TGN. COPII coats are likely to be the but has a different lipid specificity, consistent with the most ancestral, as they are used on the biosynthetic different membrane localisation. AP appendage domains pathway that will have been essential for all organisms. also recruit synaptojanin1 [48], a lipid phosphatase that The recycling back to the ER and the endocytic path- will deposphorylate the lipids in the coated vesicle to waysarelikelytobelaterspecialisations.Thestrong which many of the coat attachment proteins bind. This homology between COP1 and clathrin-AP coats implies a should make way for coat disassembly. In a similar common ancestral origin, but there is clearly a wide manner g-COP appendage domains bind ArfGAP, which divergence between these pathways. Why? One possi- stimulates the hydrolysis of Arf-GTP. Arf-GDP will bility is that there would be major advantages to being become detached from the membrane and with it the able to regulate the clathrin endocytic and recycling coat will be released from areas of metastability. ArfGAP pathways at the transcriptional level or by post-transla- activity in COPI vesicles is sensitive to membrane cur- tional modifications (principally phosphorylation and vature and thus vesicle uncoating is precisely controlled ubiquitination) independently of effects on COPI/II to occur after budding [49]. Thus there is also common- ER–Golgi trafficking. The clathrin pathway may also ality between different budding pathways at the level of be more adaptable to making larger vesicles, as illu- the accessory protein recruitment and function. Further strated by the vesicular studies of Roth and Porter on commonalities can now be explored, including the pos- mosquito oocytes [1]. It may also be that the cholesterol- sibility that clathrin-coated vesicle uncoating is also sen- and sphingomyelin-rich membrane composition of the sitive to membrane curvature. early endocytic pathways means that specialist molecules are needed. ‘Motif domains’ in accessory proteins Proteins that interact with the appendage domains do so Clathrin coats may be more versatile in their cargo selec- by short peptide motifs found in regions lacking tertiary tion and recruitment. Classical adaptors in clathrin-coated structure, which we call ‘motif domains’. Appendage vesicles have many ways to recognise cargo. The m-sub- binding motifs in these domains are often found in multi- unit is specialised to recognise YxxF motifs (see Table 1) ple copies, especially where they have a low appendage and each AP complex has a different m-subunit [50]. affinity. Thus there are eight DPW motifs (required for b subunits of AP complexes can also bind to dileucine AP2 appendage interaction) in the motif domain of motifs in cargo. Clathrin adaptors can also use other epsin1. The unstructured nature and thus greater fluidity alternative adaptors (e.g. arrestins, Dab2, GGAs and of this domain increases the chance of an individual motif Hrs) to bind to other types of cargo. By contrast, COPI finding its interaction partner. The multiple copies of coats do not use a range of alternative adaptors (although motifs and their low affinity means that these binding cargo can be bound directly to the WD40 domains of the partners have higher avidity for appendage domains when B-subcomplex); they use the equivalent of the m-subunit

Current Opinion in Cell Biology 2004, 16:379–391 www.sciencedirect.com COP and clathrin-coated vesicle budding McMahon and Mills 389

(d-COP) in the F-subcomplex to bind the accessory mediates is at a much more preliminary stage. However, protein Dsl1p, which in turn binds to cargo [51]. Similarly, even now it is clear that they are important and that the stonin 2 interacts with the m-subunit of the AP complex mechanistic basis for their formation may also be highly [52]. Dsl1p and stonin 2 alter the cargo repertoire of conserved in many trafficking pathways. The recent COPI and AP complexes through these interactions. This structureofaBARdomainandthelonglistofpredicted implies that adaptations of cargo recruitment by accessory homologues provides a pointer [35]. Tubular transport proteins may be more ancestral, with the versatility of the intermediates have high membrane curvatures at their AP m-subunit and other alternative clathrin adaptors ends and thus will have the same advantages as small being subsequent evolutionary events. vesicles in fusion reactions. Certain lipid compositions may stabilise this shape [57]. Similarly, caveoli represent Another feature of the versatility of the clathrin coat is its putative trafficking invaginations about which much ability to make flat lattices. These flat lattices are seen on remains to be discovered, but we can infer by analogy endosomes where they colocalise with Hrs and on the with other coats that caveolin polymerisation leads to plasma membrane adjacent to clathrin-coated buds cargo recruitment and membrane curvature. Caveolin [53,54]. (As a side point, this shows that clathrin poly- may even use a predicted amphipathic helix in curvature merisation itself does not drive membrane curvature, a formation in a manner akin to epsins and Arfs. point discussed from a different angle above.) What is the function of these flat lattices? They probably function to Given that evolution militates against obsolescence, sequester molecules, either keeping them from entering establishing the reasons for divergence will become the budding pathways or serving as a store or initial recruit- final challenge in completing our view of why distinct coat ment site. It is easy to envisage their formation in the complexes exist and how they work in the cell. absence of curvature-driving molecules. But how are these excluded? On the endosome the clathrin adaptor Update is Hrs [55]; significantly, this molecule has a similar Accessory proteins domain organisation to that of GGA but is missing the We have used stonin2 and Dsl1p as examples of accessory appendage domain (Figure 1), and thus will not recruit proteins interacting with components of the AP complex the epsins, amphiphysins and dynamins necessary for the (m subunit) and COPI (d) to alter cargo selection. It is now budding reaction. becoming clear that monomeric adaptors like the GGAs have their own array of accessory factors that bind to the Given the use of structurally similar appendage domains GAT (GGAs and TOM1) domain, including Rabaptin-5 in clathrin-AP complexes and in COPI b and g subunits, and TSG101. This domain also contains an Arf-binding what can we learn from the differences? There are at least site and a binding site for ubiquitin. A recent paper by 10 binding partners of the AP2 complex, six for the AP1 Mattera et al. has defined the binding site for Rabaptin-5 g-appendage, one for the AP3 complex and one for COPI in the GAT domain and the relationship between this and (Table 2). We propose that the diversity of binding the binding of ubiquitin and TSG101 [61]. This paper partners for AP2 mainly reflects the additional levels of provides a springboard to look at the relationship between regulation in this pathway. Most of the AP2 ligands were accessory proteins and the trafficking of ubiquitinated identified in brain extracts where clathrin-mediated cargo by describing the competitive and cooperative endocytosis is important for synaptic vesicle recycling. interactions between these factors and a trihelical bundle In the synapse, vesicle retrieval is stimulated by calcium in the GAT domain. It is likely that phosphorylation and activation of calcineurin, which results in the depho- ubiquitination will be a key to regulating these interac- sphorylation of many of these AP2 accessory components tions in vivo just as appears to be the case for multimeric [56]. This calcium regulation means that AP2-dependent adaptor complexes. endocytosis only occurs in response to exocytosis at the synapse. It also means that there may be partial pre- ‘Motif domains’ in adaptor proteins assembly and recruitment of endocytic proteins to endo- Bai et al. have recently reported that a sequence, WNSF, cytic zones and thus the process is primed to occur in the hinge region of GGA1 is an interaction site for the immediately following exocytosis. This diversity of acces- g ear of AP-1 [62]. This suggests that the long-reported sory proteins is absent in other coated vesicle pathways. colocalisation of GGAs and AP-1 in cells may reflect a This could reflect difficulties in detecting low-affinity and physical interaction and raises further combinatorial ques- low-abundance proteins. However, for many budding tions about cargo selection at the trans-Golgi network. pathways there may not be the necessity for these addi- Multi-hybrid yeast assays to investigate sorting signal tional layers of regulation. recognition may in future need to incorporate combina- tions of GGAs and AP-1 subunits to account for the Final remarks possibility of AP-GGA complexes. It also becomes intri- While much evolutionary conservation is apparent in guing to consider why, given this interaction, GGAs vesicle budding, the study of tubular transport inter- are often reported to be de-enriched in clathrin-coated www.sciencedirect.com Current Opinion in Cell Biology 2004, 16:379–391 390 Membranes and organelles

vesicles in contrast to AP-1 enrichment. Are GGAs both selective endocytic clathrin adaptor. EMBO J 2002, 21:4915-4926. accessory proteins for AP complexes and functional adap- 15. Pearse BM, Robinson MS: Purification and properties of 100-kd tors in their own right? proteins from coated vesicles and their reconstitution with clathrin. EMBO J 1984, 3:1951-1957. Acknowledgements 16. Dell’Angelica EC, Ohno H, Ooi CE, Rabinovich E, Roche KW, We thank Marijn Ford for transforming pdb files into structures. Bonifacino JS: AP-3: an adaptor-like protein complex with We also thank members of the lab for discussion and helpful comments. ubiquitous expression. EMBO J 1997, 16:917-928. 17. Dell’Angelica EC, Mullins C, Bonifacino JS: AP-4, a novel protein complex related to clathrin adaptors. J Biol Chem 1999, References and recommended reading 274:7278-7285. Papers of particular interest, published within the annual period of review, have been highlighted as: 18. 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factor-1 insert into membrane phospholipids upon GDP-to- 47. Miller GJ, Mattera R, Bonifacino JS, Hurley JH: Recognition of GTP exchange. Biochemistry 1997, 36:4675-4684. accessory protein motifs by the gamma-adaptin ear domain of GGA3. Nat Struct Biol 2003, 10:599-606. 33. Stenbeck G, Harter C, Brecht A, Herrmann D, Lottspeich F, Orci L, Wieland FT: b-COP, a novel subunit of coatomer. EMBO J 1993, 48. Jha A, Agostinelli NR, Mishra SK, Keyel PA, Hawryluk MJ, 12:2841-2845. Traub LM: A novel AP-2 adaptor interaction motif initially identified in the long-splice isoform of synaptojanin 1, SJ170. 34. Eugster A, Frigerio G, Dale M, Duden R: The a- and b-COP WD40 J Biol Chem 2004, 279:2281-2290. domains mediate cargo-selective interactions with distinct This paper extends the range of a-adaptin binding motifs from DxF and di-lysine motifs. Mol Biol Cell 2003. FXDXF to include a WxxF motif found in synaptojanin, AAK, GAK and stonin-2. 35. 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